This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In many smooth muscles, stretch activates contraction. However, normal filling of the urinary bladder is accomplished with minimal increase in pressure until the bladder is near functional capacity. Therefore, bladder smooth muscle must stretch and rearrange itself to allow an increase in bladder volume without pressure rise. Although a number of mechanisms are likely to be important in this response, we have recently described stretch-dependent K+ (SDK) channels in colonic smooth muscle that could hyperpolarize membrane potential and prevent activation of contraction. Recently we found SDK channels in urinary bladder. This finding stimulated the following hypotheses: 1) Functional SDK channels are present in the murine bladder and encoded by members of the two-pore family of K+ channels. 2) SDK channels mediate responses to nitric oxide. 3) Hypertrophy of the mouse bladder is accompanied by an increase in the number of functional SDK channels and may affect the performance of the detrusor muscle in these animals. 4) SDK channels are activated and inhibited by interactions with the actin cytoskeleton. In order to test these hypotheses we will use electrophysiological methods including patch-clamp studies of single SDK channels and measurement of membrane potential in intact bladder smooth muscle to investigate the role of the SDK channels in bladder function. In addition, we will use RT-PCR, immunoblot, and immunohistochemical methods to localize TREK-1 channel subunits in bladder. We will develop antisense methods and TREK- 1 knockout mouse to reduce expression or function of SDK channels in smooth muscle myocytes and in intact bladder. We will then characterize the function of these preparations to test hypotheses 1 and 2. In addition, we will implement an experimental model of bladder outlet obstruction and monitor changes in SDK channel expression (RT-PCR, immunoblot, and immunohistochemical methods) and function (electrophysiological and mechanical measurements). We will characterize the interactions between SDK channels and the actin cytoskeleton using pharmacological and antisense methods to disrupt specific interactions and examining the effects on SDK channel function. The specific actin-binding proteins associated with TREK-1 will be identified with assistance from the Cell to Proteomics Interface Core. In conclusion, the characterization of SDK channels channels in bladder will be important to understand the physiological filling mechanisms and the pathological distension.